PREreview of Tardigrade CAHS Proteins Act as Molecular Swiss Army Knives to Mediate Desiccation Tolerance Through Multiple Mechanisms

Tardigrades can survive extreme desiccation which allows them to tolerate various stresses like high (or low) temperatures and pressures, starvation, exposure to ionizing radiation etc. Unlike other desiccation tolerant organisms, tardigrades do not use or even have trehalose and sucrose in significant amounts. A group of disordered proteins were recently discovered in tardigrades, which were unique to them and were over-expressed during desiccation. These proteins could also impart desiccation tolerance when expressed in other organisms. Based on their locations, they were divided into 3 families - cytoplasmic abundant heat soluble (CAHS) proteins, mitochondrial abundant heat soluble (MAHS) proteins and secreted abundant heat soluble (SAHS) proteins. This study looks at how CAHS D helps protect other proteins during desiccation.

They observed that CAHS D could reversibly form a gel at high concentrations, which dissolved upon increasing temperature and re-gelled on decreasing temperature. Bioinformatic analysis predicted that almost the entirety of CAHS D to be disordered, with the two ends having a slight propensity of beta-sheet formation while the linker joining them was predicted to be helical. This dumbbell-like structure was confirmed with CD spectroscopy and simulations. They hypothesized that the two termini could act as stickers (forming intermolecular interactions) while the disordered linker could act as spacers (preventing intramolecular interactions) during CAHS D gelation. To test the validity of this “stickers-and-spacers” model, they tested the gel formation by CAHS D variants having different types of termini and linker lengths. Only those variants with a dumbbell-like structure with heterotypic termini could form gels, and variants with longer linkers could form gels at lower concentrations. To test if gel formation was sufficient to protect client proteins during desiccation, they performed an unfolding assay (with lactate dehydrogenase) and an aggregation assay (with citrate synthase) using each variant of CAHS D.

The gel-forming variants were the most effective in protecting lactate dehydrogenase during desiccation. The key finding of the study was that unfolding protection of each variant correlated very well to its water coordination ability per molecular mass and thus, CAHS D probably stabilized client proteins during desiccation by preserving a layer of water around the client to maintain its hydrogen bond network. But surprisingly, they found that the gel-forming variants performed the worst in protecting citrate synthase from aggregating. The variants consisting mostly of linkers were the most effective in preventing citrate synthase aggregation, as they could act as “spacers” to reduce the encounters among different citrate synthase molecules. In contrast, a gel would trap citrate synthase monomers near each other and enable aggregation instead. Hence, the authors suggest that during the early stages of desiccation, when aggregation protection is more important and CAHS D concentration is low, it functions primarily as a “spacer”, preventing aggregation-prone proteins from interacting with each other. As more water is lost and proteins begin to unfold, CAHS D forms a gel, shifting its function from being a spacer to coordinating water around proteins embedded within the gel.

Overall, the paper is nicely written and the results described are very interesting. It would be great if the authors can elaborate further on the following points.

• How do the NLN, CLC and N-terminus only variants of CAHS D coordinate water better than wild-type CAHS D despite not forming gels?

• The unfolding and the aggregation protection assays found the NLN variant of CAHS D to have a lower PD50 for both unfolding and aggregation protection. So why have not tardigrades evolved to use an NLN-type CAHS D? Does CAHS D have other roles, in which the NLN variant would be less effective? It would be interesting to observe the ability of NLN CAHS D to protect against desiccation stress in vivo.

• Unfolding protection by CAHS D and its variants was attributed to their ability to coordinate water per kDa of protein. Thus, the 2X linker version of CAHS D had improved unfolding protection than wild-type since it could form gels with larger pores, effectively coordinating more water. In contrast, the aggregation protection was attributed to the ability of CAHS D and its variants to act as “spacers” and the FL-Proline variant was best at aggregation protection. In this model, what would be the effect of spacer length and would a FL-Proline with 2X-linker variant of CAHS D better protect from aggregation compared to FL-Proline?

• Since wild-type CAHS D performs quite well in offering unfolding protection but not aggregation protection, might there be other CAHS proteins specialized/optimized for aggregation protection in tardigrades?